William Harel

Increased effectiveness of interferon alfa-2b following active specific immunotherapy for melanoma.

PURPOSE To determine whether interferon alfa-2b (IFN-alfa; intron-A, Schering Corp, Kenilworth, NJ) can induce a remission in patients previously treated with active specific immunotherapy (therapeutic melanoma vaccine) without response. PATIENTS AND METHODS Eighteen patients with disseminated melanoma who had failed to respond to at least five injections of Melacine therapeutic melanoma vaccine (Ribi ImmunoChem Research, Inc, Hamilton, MT) were then treated IFN-alfa after a 4-week interval. IFN-alfa 5 or 6 x 10(6) U/m2 was self-administered three times a week subcutaneously by melanoma patients for at least 2 months. Computed tomographic (CT) scans of the chest, abdomen, and pelvis and magnetic resonance imaging of the brain were performed within 4 weeks before treatment as a baseline, and then at 2-month intervals during treatment to evaluate response. All 18 patients were HLA-typed before treatment. The frequency of cytolytic T-cell precursors (pCTL) in the blood had been measured weekly in 13 of the patients during treatment with Melacine. RESULTS Eight of 18 patients (44.4%) had a major objective clinical response induced by IFN-alfa, including site-specific complete remissions in five. Responses lasted a median of 11 months. The median survival duration of the responders has not been reached, and exceeds 32 months. The group as a whole had a median survival duration of 10.1 months, and nonresponders lived 7.3 months. Cytolytic T-cell precursors had been increased by immunization in all five responding patients tested, but also in five of eight nonresponders. There was no association of response to IFN-alfa with specific HLA phenotypes, in contrast to our previous results with melanoma theraccine alone. CONCLUSION These data suggest an additive effect of active specific immunotherapy and IFN-alfa on the objective response rate, perhaps through upregulation of HLA molecules and tumor-associated antigens on the tumor cell by IFN-alfa, after immunization of the patient by Melacine. This treatment may have improved survival over that expected in metastatic melanoma.

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4+ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as “melanoma-specific”. Optimal demonstration of the lytic activity of CD4+ CTL required a 16-h incubation period and an effector∶target cell ratio of 40∶1. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN)γ consistently augmented lysis by these CD4+ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4+ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFNγ treatment. Thus, IFNγ may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4+CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8+ counterparts.

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

SummaryTo study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4+ and CD8+ T cell subsets. To analyze this reactivity, sorter-purified CD4+ and CD8+ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4+ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8+ grew vigorously and 29 cytolytic CD8+ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8+ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8+ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8+ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8+ and 2 CD4+ CD8+ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) Vα and Vβ gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the Vα17/Vβ7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the Vα17/Vβ7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8+ CTL. CTL with a range of melanoma specificities and different TCR αβ dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8+ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.

Efficacy of Low-dose Cyclophosphamide and Interleukin-2 in Melanoma

ABSTRACT We have studied the effects of low-dose recombinant Interleukin-2 (IL-2) preceded by low-dose cyclophosphamide on malignant melanoma. Thirty eight outpatients, with ages ranging from 25–75 years, were treated with IL-2, 3.6 million Cetus units/m2 i.v., daily for 5 days on 2 successive weeks beginning 3 days after 350 mg/m2 of i.v. cyclophosphamide. This schedule was repeated at least twice more with a 1-week interval between cycles, usually at the same dosage level. Ten of the 38 patients (26.3%) had a clinically significant remission: 2 complete (5.3%), 7 partial (18.4%), and 1 ongoing, long-term (>18 mo) “minor” response (2.6%). Four others (10.5%) had shorter minor responses and four (10.5%), a mixed response. One patient with disease restricted to the skin had a complete remission, while the other patient with a complete remission had had three lung nodules and an enlarged hilar lymph node. It was gratifying that one of the major sites of disease responding to treatment was the liver. Two complete and 2 partial remissions (i.e., > 50% regressions for > 4 wk at this site) were obtained in 10 patients with liver involvement. Lung metastases also responded, in 4 of 16 patients (1 complete and 3 partial remissions). Subcutaneous nodules responded in 7 of 21 patients (2 complete, 5 partial remissions), while lymph node metastases diminished significantly in 4 of 14 patients (1 complete, 3 partial remissions). The median duration of response was 9 months (range, 1.5-20 months), with 4 patients treated for more than one year. Toxicity was moderate and controllable, and only two patients required hospitalization, both overnight. LAK cell activation was induced in 24 of 38 patients, including all 9 of the major responders. Conversely, none of 14 patients without LAK activation had a significant clinical remission. This regimen appeared to be as effective in melanoma as those involving ex vivo activation of LAK cells, and was more tolerable than therapy with high doses of IL-2.

The Immunological Basis of Active Specific Immunotherapy (ASI) for Human Melanoma

Over the past 6 years we have studied a therapeutic vaccine (“theraccine”) for human melanoma, while at the same time using this system as an immunological model with which to learn about the mechanisms underlying rejection of a human tumor. We have also attempted to discover which melanoma-associated antigens (MAA) are immunogenic in humans, i.e., which epitopes stimulate human T helper cells, and which epitopes are targets for cytolytic T lymphocytes.

Kombination von Zytokinen mit aktiv-spezifischer Immuntherapie — Prävention von zerebralen Melanommetastasen im Mausmodell

Die Entwicklung von Hirnmetastasen bei Patienten mit metastasiertem malignen Melanom, die unter aktiv-spezifischer Immuntherapie eine Tumorremission zeigten, bereitet ein besonderes therapeutisches Problem [1]. Es ist bisher nicht bekannt, ob die Immunisierung gegen Tumoren das Zentralnervensystem, z.B. aufgrund der Blut- Hirn-Schranke, nicht erreicht, oder ob lokale Faktoren (Suppressorfaktoren, Mangel an Zytokinen, verminderte Antigenprasentation) die Immunantwort aktivierter T- Lymphozyten und Makrophagen verhindert. In dem von uns entwickelten Mausmodell zur aktiv-spezifischen Immuntherapie des Melanoms konnten wir in den Randbereichen der zerebralen Melanommetastasen CD4- und CD8-positive T-Lymphozyten sowie melaningefullte Makrophagen nachweisen. Auserdem zeigte sich ein protektiver Effekt des Melanomvakzins gegenuber Hirnmetastasen [2]. Es stellte sich daher die Frage, ob der Effekt des Vakzins durch Kombination mit zerebral aktiven Zytokinen [3], wie Interleukin-1 (IL-1), Interferon-gamma (IFNg) und Tumor-Nekrose-Faktor- alpha (TNFa) verstarkt werden kann.

Aktiv-spezifische Immuntherapie bei soliden Tumoren: Ergebnisse am Mausmodell beim malignen Melanon

Aktiv-spezifische Immuntherapie (ASI) bedeutet die Anwendung tumorassoziierter Antigene, um in einem Organismus eine Immunisierung gegen einen Tumor zu erzeugen. Dieser experimentelle onkologische Therapieansatz wird von uns mit einem aus Zelly- sat hergestellten Melanom-Vaccin und einem unspezifischen Immunstimulans (DETOX) an Patienten mit metastasierten malignen Melanomen untersucht [1]. Die Beobachtung, das aktiv-spezifische Immuntherapie bei einigen Patienten, die sich in Tumorregression befanden, letztlich letale Hirnmetastasen nicht verhindern konnte [2], fuhrte zur Entwicklung eines Melanom-Mausmodells, an dem das Metastasierungsmuster und neue Therapiemoglichkeiten untersucht werden sollten.